After the initial tumorigenic events triggered by genetic mutations of oncogenes and tumor suppressor genes occurring within tumor cells, tumor-host interactions remain as an intrinsic property for each of the critical steps leading to cancer disease progression. Growth and metastasis of solid neoplasms require the recruitment of a supporting tumor stroma, the connective tissue framework. The stromal compartment of a tumor comprises a variety of host cells, including endothelial cells, fibroblasts, and inflammatory cells. It is becoming increasingly appreciated that these host-derived cells infiltrate into tumor tissue, interact with tumor cells, and are subsequently conscripted by tumor cells to produce an array of soluble and insoluble factors that stimulate tumor angiogenesis, growth, and metastasis. These factors include integrins and cell adhesion molecules, extracellular matrix metalloproteinase inducer, as well as fibroblast activation protein (FAP, also known as seprase) that mediate the cross-talking between tumor cells and “hijacked” host stromal cells (Yan et al (2004) Preclinica 2(6):422-426). A highly consistent trait of tumor stromal fibroblasts in most epithelial cancers is the induction of FAP, a member of the serine protease family. Tumor-associated stromal cells can promote epithelial tumorigenesis, suggesting that stromal proteins may represent novel therapeutic targets (Bhowmick et al (2005) Current Opinion in. Genetics and Development 15:97-101; Joyce, J. A. (2005) Cancer Cell 7:513-520).
FAP is a cell surface serine protease expressed at sites of tissue remodeling in embryonic development. FAP is not expressed by mature somatic tissues except activated melanocytes and fibroblasts in wound healing or tumor stroma. FAP expression is specifically silenced in proliferating melanocytic cells during malignant transformation (Ramirez-Montagut et al (2004) Oncogene 23(32):5435-5446). FAP belongs to the prolyl peptidase family, which comprises serine proteases that cleave peptide substrates after a proline residue (Rosenblum et al (2003) Current Opinion in Chemical Biology 7(4):496-504; Sedo et al (2001) Biochimica et biophysica acta 1550(2):107-116; Busek et al (2004) Intl. Jour. of Biochem. & Cell Biol. 36:408-421). The prolyl peptidase family also includes dipeptidyl peptidase IV (DPP IV; also termed CD26), DPP7 (DPP II; quiescent cell proline dipeptidase), DPP8, DPP9, and prolyl carboxypeptidase (PCP; angiotensinase C). More distant members include prolyl oligopeptidase (POP or prolyl endopeptidase (PEP); post-proline cleaving enzyme; Ito, K. et al (2004) Editor(s): Barrett, Rawlings, Woessner, Handbook of Proteolytic Enzymes (2nd Edition) 2:1897-1900, Elsevier, London, UK; Polgar, L. (2002) Cellular and Molecular Life Sciences 59, 349-362) and acylaminoacylpeptidase (AAP; acylpeptide hydrolase (APH)). Proline peptidases and related proteins contain both membrane-bound and soluble members and span a broad range of expression patterns, tissue distributions and compartmentalization. These proteins have important roles in regulation of signaling by peptide hormones, and are emerging targets for diabetes, oncology, and other indications.
FAP (seprase) was isolated from bovine serum, purified to homogeneity, and sequenced (Collins et al (2004) Intl. Jour. of Biochem. & Cell Biol. 36(11):2320-2333). The protease activity of bovine FAP in cleaving synthetic peptide substrates suggests that: (i) multiple subsites in FAP are involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide; (ii) there is high primary substrate specificity for the Pro-X bond; and (iii) there is a preference for a hydrophobic residue at the C-terminal end of the scissile bond.
It was demonstrated that FAP has both dipeptidyl peptidase and collagenolytic activity capable of degrading gelatin and type I collagen. The expression and enzyme activity of FAP in benign and malignant melanocytic skin tumors has been established, indicating a possible role for FAP in the control of tumor cell growth and proliferation during melanoma carcinogenesis (Huber et al (2003) Jour. of Investigative Dermatology 120(2):182-188), colorectal cancer (Satoshi et al (2003) Cancer letters 199(1):91-98), and breast cancer (Goodman et al (2003) Clinical & Exp. Metastasis 20(5):459-470), as well as all of breast, colon, and lung cancer (Park et al (1999) J. Biol. Chem. 274:36505-36512). Furthermore, FAP seems to upregulated in cirrhosis (Levi, M T et al (1999) Hepatology 29:1768-1778), fibromatosis (Skubitz, K M et al J. Clin. Lab. Med. (2004) 143(2):89-98), and rheumatoid arthritis.
The fibroblast activation protein alpha (FAPα) was discovered with a monoclonal antibody, mAb F19, that was generated in the course of a serological survey of cell surface antigens expressed on cultured human fibroblasts, sarcomas and neuroectodermal tumor cells. This antibody was used to characterize the plasma membrane-associated 95 kDa FAPα glycoprotein, to isolate the FAP-encoding cDNA, and to examine FAPα expression in a broad range of normal and neoplastic human tissues (Park, John E.; Rettig, Wolfgang J., Editor(s): Barrett, Alan J.; Rawlings, Neil D.; Woessner, J. Fred, Handbook of Proteolytic Enzymes (2nd Edition) (2004) 2:1913-1917, Publisher: Elsevier, London, UK).
Maturation of blood cells via hematopoiesis involves cytokines and their regulation by the serine proteases CD26/dipeptidyl-peptidase IV (DPP-IV), as well as FAP (McIntyre et al (2004) Drugs of the Future 29(9):882-886; Ajami et al (2003) Biochemistry 42(3):694-701). The human fibroblast activation protein (FAPα) is a Mf 95,000 cell surface molecule originally identified with monoclonal antibody (mAb) F19 (Rettig et al. (1988) Proc. Natl. Acad. Sci. USA 85, 3110-3114; Rettig et al. (1993) Cancer Res. 53, 3327-3335; Rettig et al (1994) Intl. Jour. of Cancer 58(3):385-392). The FAP gene, localized to chromosome 2 in humans (Mathew et al (1995) Genomics 25(1):335-337) is a 2812 nt sequence with an open reading frame of 2277 bp conserved throughout a variety of species including mouse, hamster, and Xenopus laevis (Scanlan et al (1994) Proc. Natl. Acad. Sci. USA 91:5657-5661; Park et al (1999) J. Biol. Chem. 274:36505-36512; Niedermeyer et al (1998) Eur. J. Biochem. 254:650-654). The corresponding FAP protein product contains 759 or 760 amino acids and has a calculated molecular weight of about 88 kDa. The primary amino acid sequence is homologous to type II integral membrane proteins, which are characterized by a carboxy-terminal end that is large and corresponds to the extra-cellular domain (ECD), a hydrophobic transmembrane segment, and a short cytoplasmic tail. FAP is highly homologous to dipeptidyl peptidase IV (DDPIV) in various species, with 61% nucleotide sequence identity and 48% amino acid sequence identity to DPPIV. Although both FAP and DDPIV have peptidase (protease) activity, biochemical and serological studies show that these proteins are significantly different in their enzymatic activity with synthetic substrates as well as their functional activation of T lymphocytes (DDPIV induction) or reactive stromal fibroblasts (FAP induction (Mathew et al (1995) Genomics w5:335-337). The FAPα cDNA codes for a type II integral membrane protein with a large extracellular domain, trans-membrane segment, and short cytoplasmic tail (Scanlan et al. (1994) Proc. Natl. Acad. Sci. USA 91, 5657-5661; U.S. Pat. No. 6,846,910; WO 97/34927; U.S. Pat. No. 5,767,242; U.S. Pat. No. 5,587,299; U.S. Pat. No. 5,965,373). FAPα shows 48% amino acid sequence identity to the T-cell activation antigen CD26, also known as dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5), a membrane-bound protein with dipeptidyl peptidase activity. FAPα has enzymatic activity and is a member of the serine protease family, with serine 624 being critical for enzymatic function WO 97/34927; U.S. Pat. No. 5,965,373). Seprase (FAPα) is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX (Pineiro-Sanchez et al (1997) Jour. of Biol. Chem. 272(12):7595-7601), and which promotes rapid tumor growth in a mouse model of human breast cancer (Huang et al (2004) Cancer Res. 64:2712-2716). Molecular cloning of a cDNA encodes the 97 kDa subunit of seprase with a deduced amino acid sequence that predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein α (FAPα) are products of the same gene and are essentially identical (Goldstein et al (1997) Biochimica et Biophysica Acta 1361(1):11-19).
FAPα is selectively expressed in reactive stromal fibroblasts of many histological types of human epithelial cancers, granulation tissue of healing wounds, and malignant cells of certain bone and soft tissue sarcomas. Normal adult tissues are generally devoid of detectable FAPα (Chen et al (2003) Adv. Exp. Med. Biol. 524:197-203), but some fetal mesenchymal tissues transiently express the molecule. In contrast, most of the common types of epithelial cancers, including >90% of breast, non-small-cell lung, and colorectal carcinomas, contain FAPα-reactive stromal fibroblasts. These FAPα+ fibroblasts accompany newly formed tumor blood vessels, forming a distinct cellular compartment interposed between the tumor capillary endothelium and the basal aspect of malignant epithelial cell clusters (Welt et al. (1994) J. Clin. Oncol. 12(6), 1193-1203). While FAPα+ stromal fibroblasts are found in both primary and metastatic carcinomas, the benign and premalignant epithelial lesions tested, such as fibroadenomas of the breast and colorectal adenomas, only rarely contain FAPα+ stromal cells. Based on the restricted distribution pattern of FAPα in normal tissues and its uniform expression in the supporting stroma of many malignant tumors, clinical trials with 131I-labelled mAb F19 have been initiated in patients with metastatic colon carcinomas (Tanswell et al (2001) British Jour. of Clin. Pharm. 51(2):177-180). Evidence for the promotion of tumor growth by murine FAP, and inhibition of tumor growth by antibody inhibitors of FAP was demonstrated by Cheng et al (2002) 62:4767-4772. Human FAP was expressed and targetted by 131I-labelled humanized anti-FAP mAb in a human skin/severe combined immunodeficient mouse breast cancer xenograft model (Tahtis et al (2003) Molecular Cancer Therapeutics 2(8):729-737).
A high-resolution X-ray crystal structure of the extracellular domain of FAPα revealed a difference from DPP-IV in their active sites. Kinetic analysis of an active site mutant of FAPα, A657D, with dipeptide substrates showed an increase in the rate of cleavage for a free amino terminus substrate but a decrease for the corresponding N-benzyloxycarbonyl substrate, relative to wild type FAPα (Aertgeerts et al (2005) J. Biol. Chem., April; 10. 1074/jbc.C500092200).
Four completely human antibody derivatives (single-chain-antibody fragments, scFvs) with specificity for FAP as a general tumor stroma marker were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy-chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors (Schmidt et al (2001) European Jour. of Biochemistry 268(6):1730-1738). Other recombinant FAP-binding proteins with framework modifications have been expressed (U.S. Pat. No. 6,455,677). Although attempts to fully block FAP activity with antibodies have not been successful (Cheng, et al (2004) Abrogation of Fibroblast Activation Protein Enzymatic Activity Attenuates Tumor Growth. In. American Association for Cancer Research, 95th Annual Meeting, Orlando, Fla.), Sibrotuzumab, a humanized monoclonal antibody directed against FAP, is in human clinical trials for cancer therapy (Kloft et al (2004) Investigational New Drugs 22(1):39-52; Scott et al (2003) Clinical Cancer Research 9(5): 1639-1647; Cheng et al (2003) Clinical Cancer Research 9(5):1590-1595; Hofheinz et al (2003 February) Onkologie 26(1):44-8).
The amino boronic dipeptide (talabostat, PT-100, Val-boro-Pro; Point Therapeutics), a dipeptidyl peptidase (DPP) inhibitor, has been shown to up-regulate gene expression of certain cytokines in hematopoietic tissue via a high-affinity interaction, which appears to involve fibroblast activation protein (US 2003/0158114; US 2004/0152192; Adams et al (2004) Cancer Research 64(15):5471-5480; Jones et al (2003) Blood 102(5):1641-1648). Because FAP is also expressed in stroma of lymphoid tissue and tumors, the effect of PT-100 on tumor growth was studied in mice in vivo although PT-100 has no direct cytotoxic effect on tumor cells in vitro. Oral administration of PT-100 to mice slowed growth of syngeneic tumors derived from fibrosarcoma, lymphoma, melanoma, and mastocytoma cell lines. Treatment of mice with PT-100 resulted in tumor growth attenuation in a tumor model characterized by murine FAP expression in the surrounding tumor stromal fibroblasts (Cheng et al (2005) Mol. Cancer Ther. 4(3):351-60). However, PT-100 is not FAP-specific because it also inhibits DPP-8 and DPP-9. In addition, PT-100 demonstrates a self-inactivation mechanism by intramolecular cyclization of the N-terminus amine and the boronate group. A phase I/II human clinical study has been initiated to test the safety and efficacy of talabostat in combination with RITUXAN® (Genentech, Inc.) in patients with hematologic malignancies, such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Other inhibitors targeting prolyl peptidases, include: Val-BoroPro compounds (Flentke et al (1991) Proc. Natl. Acad. Sci. USA 88:1556-1559; Coutts et al (1996) J. Med. Chem. 39:2087-2094; Shreder et al. (2005) Bioorganic and Medicinal Chemistry Letters 15:4256-4260); N-acyl-Gly-BoroPro compounds (Edosada et al (2006) Jour. Biological Chem. 281(11):7437-7444); N-alkyl-Gly-BoroPro compounds (Hu, et al (2005) Bioorganic and Medicinal Chemistry Letters 15:4239-4242); 1-(2′-aminoacyl)-2-cyanopyrrolidine compounds (WO 2001/040180); and Boro-norleucine compounds (Shreder et al (2005) Bioorganic and Medicinal Chemistry Letters 15:4256-4260).
Peptidic prodrugs which are FAP cleavage substrates have been reported to be converted to cytotoxic or cytostatic metabolites by the sequence selective cleavage of FAP (U.S. Pat. No. 6,613,879; US 2003/021979; US 2003/0232742; US 2003/0055052; US 2002/0155565). Peptide proline-boronate protease inhibitors have been reported (Bachovchin et al (1990) Jour. Biol. Chem. 265(7):3738-3743; Flentke et al (1991) Proc. Natl. Acad. Sci. 88:1556-1559; Snow et al (1994) J. Amer. Chem. Soc. 116(24):10860-10869; Coutts et al (1996) J. Med. Chem. 39:2087-2094; U.S. Pat. No. 4,935,493; U.S. Pat. No. 5,288,707; U.S. Pat. No. 5,462,928; U.S. Pat. No. 6,825,169; WO 2003/092605; US 2004/0229820; WO 2005/047297). Cyclic boro-proline compounds are reported to be useful for oral administration (U.S. Pat. No. 6,355,614). An N-acetyl lysine proline boronate compound has been proposed as an antibacterial agent (U.S. Pat. No. 5,574,017).